The process of introducing DNA (and other similar polynucleotides) into host cells is a key aspect of recombinant DNA technology. The process by which polynucleotides are introduced into host cells is called transformation. Bacterial cells generally remain the preferred hosts for the majority of recombinant DNA experiments and genetic engineering manipulations. Of particular interest for genetic engineering experiments is the bacteria Escherichia coli. Given that “competence” (the ability to efficiently uptake exogenous DNA) is not a natural feature of the E. coli growth cycle, artificial procedures must be used to introduce exogenous polynucleotides into E. coli. Of particular interest, are a variety of competency inducing procedures that render bacteria, including E. coli, more permeable to exogenous nucleic acid. Bacterial cells that have been treated to enhance their permeability to polynucleotides are generally referred to as competent cells.
There are many established procedures for making competent cells. These procedures include the CaCl2 incubation methods of Mandel and Higa, J. of Mol. Biol. 53:159 (1970), as well as numerous well-known variants thereof. Hanahan has made a detailed study of factors that effect the efficiency of transformation of E. coli cells (J. Mol. Biol. 166:557–580 (1983)) where he describes a method of producing highly competent E. coli cells comprising the step of washing E. coli cells in a buffer comprising potassium acetate, KCl, MnCl2, CaCl2, and hexamine cobalt chloride, which is generally regarded as the best available method of producing highly competent E. coli. Another method of producing competent E. coli cells is described by Jessee et al., U.S. Pat. No. 4,981,797. Jessee et al. shows that high levels of competency may be induced by growing E. coli cells in a temperature range of 18° C. to 32° C. as part of the competency inducing procedure.
The various techniques for rendering E. coli cells competent produce competent E. coli cells having varying of transformation efficiencies. The precise mechanism by which DNA enters competent E. coli is not completely understood. Nor is it completely understood why one composition of competent E. coli cells differs in transformation efficiency from that of another composition of competent E. coli cells. Hanahan, in Escherichia Coli and Salmonella Typhimurium: Cellular and Molecular Biology, editor F. C. Neidhardt, American Society for Microbiology, Washington, D.C. (1987).
The above methods have been further optimized to achieve efficiencies of approximately 1×109 cfu/μg supercoiled plasmid DNA. Although this number appears high, the theoretical efficiency for the test plasmid (pUC) is 3×1011 cfu/μg. Furthermore, when applied to practical laboratory conditions, such as the transformation of DNA substrates that were ligated rather than supercoiled, the actual number of colony forming units observed was many orders of magnitude lower than that achieved when supercoiled pUC was used as a test substrate.
Even with past developments, only a minute fraction of the cells in a preparation of “competent” E. coli cells, are actually competent for DNA uptake. Thus, the methods and cells presently used to generate compositions of competent E. coli cells may yet be significantly improved.
Alternatively, other methods of producing competent cells may result in the formation of competent E. coli cells that each have an enhanced ability to replicate and expresses exogenously added DNA. Hanahan, in J. Mol. Bio. 166:557–580 (1983) has speculated that competent E. coli cells contain channels for transport of DNA across the cell envelope, and that the limiting step in determining the competency for transformation of E. coli cells are events that occur in the cell after the cell has taken up the DNA of interest, i.e., the establishment step. Another factor affecting the transformation efficiency of a composition of competent E. coli cells is the genotype of the cells. Some strains of E. coli are known to produce more highly transformable competent cell compositions than other strains of E. coli that have been subjected to the same competency inducing procedure.
Subsequent to the initial discovery that E. coli could be rendered competent for DNA uptake, studies have been undertaken to increase transformation efficiency. The maximum level of transformation efficiency obtained using the method of Hanahan described in J. Mol. Bio. 166:557–580 (1987), which employs the step of washing cells in a buffer comprising potassium acetate, KCl, MnCl2, CaCl2, glycerol, and hexamine cobalt chloride, is approximately 1×109 transformants per microgram of supercoiled pUC18 plasmid DNA. On a per cell basis, this translates to approximately 1 cell out of 300 in the population actually becoming transformed. However, the above number generally only applies to small supercoiled plasmids. When large plasmids, or ligated molecules are involved, as is the case with many recombinant DNA experiments, the number of “competant” cells that actually become transformed is dramatically reduced. As such, a need continues to exist for new and improved methods for producing competent E. coli of superior transformability, as well as new strains of E. coli that demonstrate superior transformability. Such methods and strains would be of wide interest to most researchers in the field of genetic engineering in that the number of transformations required to obtain the desired result would be minimized. Thus, for example, larger genetic libraries could be built more easily as well as the construction of complex recombinant molecules achieved more readily.